Site directed maleimide bifunctional chelators for the M(CO) 3+ core (M = 99mTc, Re)

Article abstract of DOI:10.1039/b417588c

A series of bifunctional chelates containing a tridentate donor set for complexation of the M(CO)₃⁺ core and a maleimide group for site-specific coupling to peptides and proteins containing free thiol groups has been prepared and their Re(CO)₃⁺ complexes and glutathione conjugates structurally characterized. The flexibility of design allows preparation of ligands suitable for both fluorescence imaging, radioimaging and radiotherapeutic studies of proteins and peptides as well as other biopolymers using site specific conjugation. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b417588c

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+
Site directed maleimide bifunctional chelators for the M(CO)3 core
(M 5 99mTc, Re){
Sangeeta Ray Banerjee,a John W. Babichb and Jon Zubieta*a
Received (in Cambridge, UK) 19th November 2004, Accepted 21st January 2005
First published as an Advance Article on the web 7th February 2005
DOI: 10.1039/b417588c
PEG conjugation of proteins have been described in which PEG is
activated with a maleimide group capable of specifically forming a
covalent bond with free thiols on the surface of proteins.9 For
these reasons, maleimide linkers10 are of particular interest to
bioconjugate chemistry as the stoichiometry and site attachment
are predictable and significant alterations in biological activity may
be avoided. In this study, we report the syntheses and
characterizations of novel maleimide-dpa and maleimide-dqa
(dpa 5 dipicolylamine; dqa 5 diquinolinoyl methylamine)
A series of bifunctional chelates containing a tridentate donor
+
set for complexation of the M(CO)3 core and a maleimide
group for site-specific coupling to peptides and proteins
containing free thiol groups has been prepared and their
+
Re(CO)3 complexes and glutathione conjugates structurally
characterized. The flexibility of design allows preparation of
ligands suitable for both fluorescence imaging, radioimaging
and radiotherapeutic studies of proteins and peptides as well as
other biopolymers using site specific conjugation.
+
derivatives for 99mTc labeling of peptides and of their Re(CO)3
complexes.
The significant development in recent years of the chemistry of the
Group 7 congeners technetium and rhenium reflects the applica-
tions of the radionuclides 99mTc and 186,188Re in the development
of diagnostic and therapeutic radiopharmaceuticals, respectively.1,2
Technetium-99m is the most widely used radionuclide in diagnostic
medicine owing to its ideal nuclear properties, low cost and
widespread availability.3,4 In addition to its important radio-
isotopes, the nonradioactive isotopes of rhenium (185,187Re)
provide both model chemistry for the radioactive analogues and
materials which may serve as luminescent probes.5 We have
recently reported the synthesis of a series of M(I) binding ligands
(M 5 Tc, Re) based on a lysine derived bis(pyridyl)amine, referred
to as a single amino acid chelate (SAAC), which forms inert
complexes with the chemically robust M(CO)3+ core (M 5 99mTc,
Re). SAAC can be readily incorporated into peptides using
conventional solid phase synthetic methods as if it were a natural
amino acid.6 Furthermore, we have prepared the Re(I) complex of
the bis-quinolinyl amine analog of SAAC that exhibits attractive
fluorescence properties.7
The bifunctional chelate L1 was prepared from the reaction of
bis(picolyl)aminopropanol, prepared as previously described,6c
with maleimide using Mitsunobu conditions.10{ Appropriate
modifications allowed the synthesis of a family of bifunctional
chelates of the type maleimide-(CH2)nNRR9 [n 5 2, 3, 6,
R 5 R9 5 quinolinoylmethyl, (L2–L4); n 5 3, R 5 R9,
–CH2CO2H (L5); R 5 –CH2CO2H, R9 5 pyridylmethyl and
quinolinoylmethyl, respectively- (L6, L7)] (Scheme 1) providing a
+
range of effective tridentate donors for the M(CO)3 unit and
allowing the isolation of neutral, cationic or anionic complexes
[M(CO)3(L)]n, depending on the donor group identity. Ligand L2
has been structurally characterized after recrystallization from
chloroform as L2?HCCl3.§ Furthermore, the bifunctional chelates
L2–L4 provide ligands whose Re(I) complexes should exhibit
fluorescence.7
+
The Re(CO)3 complexes of L1–L4 were prepared by the
reaction of (NEt4)2[Re(CO)3Br3]11 with the appropriate ligand. As
shown in Fig. 1, the structure of [Re(CO)3(L3)]Br consists of
discrete [Re(CO)3(L3)]+ cations and Br2 anions.§ The Re(I) site
exhibits distorted octahedral geometry through coordination to
three carbonyl groups in a meridional arrangement, the amine
nitrogen and the nitrogen donors of the two quinoline units.
To assess the coupling of the maleimide derivatized ligands,
reactions between L1, L3, L4 and glutathione (glu-cys-gly) were
performed by reacting L and glutathione in a 1:1 molar ratio in
PBS buffer at pH 7.4, followed by reverse phase column
chromatographic (Sep-Pak) purification. Yields of greater than
We now have turned our attention to the application of this
coordination chemistry to site directed modification of peptides
and proteins. Many bifunctional chelates can be labeled with a
variety of radiometals,8 primarily through acylation of the primary
amine groups of lysine residues. While a generally useful approach,
there are significant limitations to selective conjugation of specific
amino groups in the protein or peptide of interest. Because the free
thiol functional group is not very common in most proteins or
peptides and can be labeled with high chemoselectivity, thiol-
reactive reagents provide a means of selectively modifying a
biopolymer at a defined site. Additionally, using solid phase
peptide synthesis or protein engineering, it is possible to
incorporate a cysteine residue at a defined position in peptides
and proteins, respectively. Furthermore, methods for effective
{ Electronic supplementary information (ESI) available: A: Labeling of
L1–L4 with 99mTc(CO)3+. B: Absorption and fluorescence studies. C:
X-Ray structure of L2. See http://www.rsc.org/suppdata/cc/b4/b417588c/
*jazubiet@syr.edu
Scheme 1
1784 | Chem. Commun., 2005, 1784–1786
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3MLCT [dp(Re) A p*(ligand)] transition. The measured quantum
yield of the complex (W 5 0.015) in ethylene glycol under argon
atmosphere is low but not significantly different from the
fluorescent quantum yields reported for other transition metal
based complexes currently employed as fluorescent probes.7,13
Furthermore, the fluorescence lifetime of the complex of ca. 16 ms
renders cellular imaging practical. Peptide and protein conjugates
of [Re(CO)3(L4)]+ also exhibit similar photophysical properties as
the parent complex. For example, [Re(CO)3(L4-glutathione)]+
exhibits a strong absorbance at ca. 322 nm in ethylene glycol,
giving rise to peak fluorescence intensity at 555 nm, with a lifetime
of ca. 14 ms under Ar atmosphere.
Sangeeta Ray Banerjee,a John W. Babichb and Jon Zubieta*a
aDepartment of Chemistry, Syracuse University, Syracuse, NY,
13244-4100, USA. E-mail: jazubiet@syr.edu; Fax: 315-443-4070
bMolecular Insight Pharmaceuticals, 160 Second Street, Cambridge,
MA, 02142, USA
Notes and references
{ Representative syntheses: Bis(picolyl)aminopropylmaleimide (L1): DEAD
(0.297 g, 1.71 mmol) was added dropwise over a period of 15 min
to a solution of bis(picolyl)aminopropanol (0.364 g, 1.4 mmol), PPh3
(0.437 g, 1.65 mmol) and maleimide (0.167 g, 1.72 mmol) in THF
(15 mL) at 0 uC under nitrogen atmosphere. The mixture was then
stirred at 0 uC for another 5–6 h and subsequently poured into water
and extracted with diethyl ether. The ether extract was dried over Na2SO4
and concentrated. The residue was purified through flash column
chromatography with 2% MeOH–CH2Cl2 to give the product as a
Fig. 1 A view of the structure of [Re(CO)3(L3)]Br, showing the atom-
labeling scheme and 50% probability ellipsoids.
90% of the peptide-ligand-maleimide conjugate were achieved.
Alternatively, the [Re(CO)3(L)]Br complexes could be directly
coupled to the glutathione under conditions similar to those
described above to produce the metalated conjugates (Scheme 2) in
80–90% yield.
1
yellow oil in 55% yield. H NMR (300 MHz, MeOH-d4): d 8.49 (d, 2H),
7.61 (t, 2H), 7.46 (t, 2H), 7.05 (d, 2H), 6.66 (s, 2H), 3.77 (s, 4H), 3.48
(t, 2H), 2.5 (t. 2H), 1.75 (t, 2H). 13C NMR: d 170.69, 159.57, 148.95, 136.42,
134.04, 123.05, 121.98, 60.23, 51.44, 36.04, 26.17. HRMS (1:1 MeOH–THF
+ NaCl): Calc. for C19H20N4O2Na+ m/z 359.1478; found 359.14608.
Bis(qinolinoylmethyl)aminopropylmaleimide (L3): this was prepared in a
similar fashion to L1, with bis(quinolinolymethyl)aminopropanol in place
of bis(picolyl)aminopopanol. 1H NMR (300 MHz, CDCl3): d 8.13
(d, J 5 8.4 Hz, 2H), 8.05 (d, J 5 8.4 Hz, 2H), 7.80–7.68 (m, 6H), 7.50
(t, J 5 9.0 Hz, 2H), 6.53 (s, 1H), 4.00 (s, 4H), 3.51 (t, J 5 7.2 Hz, 2H), 2.66
(t, J 5 6.9 Hz, 2H), 1.82 (m, 2H). 13C NMR (300 MHz, MeOH-d4): d
170.85, 160.44, 147.70, 136.57, 134.09, 129.54, 129.28, 127.69, 126.35,
121.39, 61.53, 51.78, 36.20, 26.36. Mp 143–145 uC. HRMS (1:1 MeOH–
THF): Calc. for C27H24N4O5Na+ m/z 459.179144; found 459.17710.
[Re(CO)3(L1)]Br: A solution of (NEt4)2[Re(CO)3Br3] (387 mg, 0.50 mmol
in 20 ml methanol) was added to a solution of L1 (168 mg, 0.50 mmol in
5 ml) and heated to reflux under an argon atmosphere for 3 h. After
cooling the reaction mixture to room temperature, the solvent was removed
under reduced pressure, and the solid residue was dissolved in 20 ml of
CH2Cl2. Extraction with water (3 6 30 ml) removed the tetraethylammo-
nium bromide. The organic layer was dried over sodium sulfate, and the
solvent volume was reduced to y1 ml. Standard chromatographic
purification on a silica gel column with 2:98 MeOH–CH2Cl2 solution
gave the purified product in 81% yield (278 mg). 1H NMR (300 MHz,
MeOH-d4): d 8.84 (d, J 5 6.0 Hz, 2H), 7.94 (t, J 5 8.1 Hz, 2H), 7.66
(d, J 5 9.0 Hz, 2H), 7.38 (t, J 5 6.0 Hz, 2H), 6.87 (s, 2H), 4.85 (dd, J 5
16.5 Hz, 4H), 3.89 (m, 2H), 3.69 (t, J 5 6 Hz, 2H), 2.25 (m, 2H). 13C NMR
(300 MHz, MeOH-d4): d 197.21, 196.59, 172.76, 162.12, 153.25 141.81,
135.78, 127.13, 125.04, 69.52, 86.85, 36.14, 26.08. HRMS (MeOH):
Calc. for C22H20N4O5Re+ m/z 607.0985; found 607.099. [Re(CO)3(L3)]Br
and [Re(CO)3(L4)]Br were prepared in an analogous fashion to
[Re(CO)3(L1)]Br. Yields: y80%. [Re(CO)3(L3)]Br: 1H NMR
(300 MHz, MeOH-d4): d 8.53 (d, J 5 8.4 Hz, 2H), 8.45 (d, J 5 8.7 Hz,
2H), 7.99 (d, J 5 9.0 Hz, 2H), 7.83 (t, J 5 9.0 Hz, 2H), 7.72–7.63 (m, 4H),
6.84 (s, 1H), 5.25 (m, 4H), 3.97 (m, 2H), 3.72 (m, 2H), 2.36 (m, 2H). 13C
NMR (300 MHz, MeOH-d4): d 197.24, 195.50, 172.64, 166.55, 148.13,
143.03, 135.67, 134.11, 131.01, 129.84, 129.53, 121.19, 70.09, 67.23, 36.08,
26.95, Mp 206–208 uC. IR(KBr): 2029, 1919, 1705, 1515. HRMS (1:1
MeOH–THF): Calc. for C30H24N4O5Re+ m/z 707.129866; found
Radiolabeling of the maleimide derivatized ligands may be
achieved by reacting the ligands with [99mTc(CO)3(H2O)3]+,12 at
90 uC for 20 min. The labeling yields were greater than 85%, with
greater than 95% radiochemical purity before HPLC purification,
based on radiochromatograms (ESI, Fig. S1{). The products
are stable for at least 24 h, and the procedure allows labeling to
1 mg ml21
.
The flexibility of the ligand design permits the introduction of
chelates, as noted for ligands L2–L4, which would allow the
preparation of fluorescent and radioactive chelate complexes,
which are isostructural. The long emission lifetime and large
Stokes’ shift of fluorescent Re complexes are particularly attractive
in this application. The complex [Re(CO)3(L4)]Br exhibits a strong
UV absorbance with a maximum at 321 nm. Upon photoexcita-
tion, long-lived fluorescence emission at 550 nm is produced with a
lifetime of 15.9 msec in ethylene glycol under Ar, assigned to a
1
707.12556.[Re(CO)3(L4)]Br: H NMR (300 MHz, CDCl3): d 8.28–8.23
(m, 4H), 7.79 (d, J 5 8.7 Hz, 2H), 7.68 (d, J 5 8.1 Hz, 2H), 7.60 (d,
Scheme 2
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J 5 8,7 Hz, 2H), 7.48 (t, J 5 6.0 Hz, 2H), 6.55 (s, 1H), 5.25 (m, 4H), 3.97
(m, 2H), 3.72 (m, 2H), 2.36 (m, 2H). 13C NMR, 300 MHz, MeOH-d4): d
197.24, 195.50, 172.64, 166.55, 148.13, 143.03, 135.67, 134.11, 131.01,
129.84, 129.53, 121.19, 70.09, 67.23, 36.08, 26.95 uC. IR (KBr): 2023, 1924,
1700, 1513. HRMS (1:1 MeOH–THF): Calc. for C33H30N4O5Re+ m/z
749.176816, found 749.17669. Coupling of [Re(CO)3(L4)]Br with
glutathione: A solution of [Re(CO)3(L4)]Br (24 mg, 0.029 mmol) in
DMF (0.7 ml) was added dropwise to a solution of glutathione (9 mg,
0.029 mmol) in phosphate-buffered saline (PBS, pH 7,5, 1 ml). The mixture
was stirred at room temperature for 40 min. At this time TLC indicated the
disappearance of [Re(CO)3(L4)]Br (Rf 0.5; MeOH–CH2Cl2 2:98) and the
appearance of one of the major product (Rf 0.6; n-BuOH–MeOH–H2O–
AcOH 4:2:1:0.5). After dilution with water (3.3 ml) the mixture was passed
through a Waters Sep-Pak C-18 cartridge (1 ml). The cartridge was washed
with water (3 6 5 ml) and the product was eluted with 2:1 MeOH–water
(5 ml). Evaporation of the eluate afforded the product as a colorless solid.
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1
Yield: 27 mg (y82%). H NMR (300 MHz, MeOH-d4): d 8.78 (m, 4H),
8.24 (d, J 5 8.7 Hz, 2H), 8.11 (t, J 5 6.9 Hz, 2H), 7.9–7.76 (m, 4H), 5.30–
4.6 (m, 5H), 3.95–3.81 (m, 3H), 3.81–3.72 (m, 2H), 3.69–3.63 (m, 1H), 3.62–
3.48 (m, 3H), 3.33–2.81 (m, 2H), 2.72–2.41 (m, 3H), 2.28–1.94 (m, 4H),
1.75–1.32 (m, 8H). HRMS (1:1 MeOH–THF + NaCl): Calc. for
C43H47N7O11ReSNa+ m/z 1079.25039 and C43H47N7O11ReSH+ m/z
1057.268448, found 1079.24893 and 1057.27396.
¯
§ Crystal data: L2?HCCl3, C27H23Cl3N4O2, triclinic, P1, a 5 9.2492(8),
˚
b 511.175(1), c 5 13.096(1) A, a 5 101.983(2), b 5 90.707(2),
3
c 5 101.991(2)u, V 5 1293.1(2) A , Z 5 2, Dc 5 1.392 g cm23; structure
˚
solution and refinement converged at R1 5 0.0577 and wR2 5 0.1250 for
8452 reflections (all data to h 5 31.50u; ESI, Fig. S4). [Re(CO)3(L3)]Br,
¯
C30H24BrN4O5Re, triclinic, P1, a 5 11.639(2), b 5 11.829(2), c 5
3
˚
˚
13.249(3) A, a 5 65.300(3), b 5 77.198(3), c 5 72.582(3)u, V 5 1571.3(5) A ,
Z 5 2, Dc 5 1.663 g cm23; structure solution and refinement converged at
R1 5 0.0602 and wR2 5 0.1299 for 10184 reflections (all data to h 5 31.50u).
CCDC 253367 and 258469. See http://www.rsc.org/suppdata/cc/b4/
b417588c/ for crystallographic data in .cif or other electronic format.
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1786 | Chem. Commun., 2005, 1784–1786
This journal is ß The Royal Society of Chemistry 2005